PROJECT SUMMARY The goal of this Mentored Clinical Scientist Research Career Development Award (K08) is to provide a 5-year training pathway for Anthony Orvedahl, MD, PhD, to become an independent investigator. Dr. Orvedahl obtained the MD, PhD degree in the Medical Scientist Training Program at the UT Southwestern Medical Center, where he studied the role of autophagy in innate antiviral immunity. After training in Pediatrics and Infectious Diseases at St. Louis Children's Hospital/ Washington University, he joined the lab of Herbert ?Skip? Virgin, MD, PhD, who is a renowned expert in antiviral immunity. Gary Silverman, MD, PhD, who will serve as the primary Co-Mentor, is a practicing physician-scientist in Neonatology with recognized expertise in mechanisms of cell death. In his preliminary work using genome-wide CRISPR screening, Dr. Orvedahl identified an important role for autophagy genes (including Atg5) in regulating IFN?-induced cell death. A suppressor CRISPR screen on an Atg5-deficient background revealed the TNF pathway as a mediator of the hypersensitivity to cell death. However, TNF was insufficient on its own to trigger cell death, which indicated one or more additional IFN?-induced factors contribute to TNF-induced death. Preliminary work suggests that one of these factors is the IFN?-induced mitochondrial enzyme, immune regulated gene 1 (IRG1). Importantly, mice with myeloid cell-specific autophagy gene- deficiency exhibited markedly decreased survival in a TNF-induced model of shock. However, the specific type of cell death induced by IFN? in combination with TNF, the mechanism of IRG1 and its enzymatic product itaconate, and the in vivo role of these factors during TNF-induced shock remain unclear. The proposed studies to address these questions in this K08 will require in-depth analyses of cell death and mitochondrial homeostasis using multiple microscopic, flow cytometric, and biochemical techniques. Washington University School of Medicine provides an ideal environment to pursue the training plan outlined in this proposal. The Department of Pediatrics has a longstanding commitment and track record of training independent physician-scientists. Dr. Orvedahl has established successful collaborations related to this project, and interacts frequently with investigators in the departments of Pediatrics and Pathology/ Immunology. An oversight committee has been formed that includes advisors with expertise in immunometabolism, autophagy in intestinal inflammation, protein biochemistry, and macrophage immune responses. The training plan incorporates technical workshops, as well as formal coursework on grantsmanship, which will facilitate transition to independence towards the end of the award period. The combination of mentorship, institutional resources, and focused training activities offer an unparalleled opportunity to achieve the goals of this proposal. The immediate research goals described herein are to: 1) investigate the mechanism of autophagy in protection against cytokine-induced macrophage cell death; 2) evaluate the role of a novel factor, IRG1, that links mitochondria to the mechanism of cell death; and 3) apply these findings in vivo to a model of fatal TNF-induced shock.